Jonathan D. Geiger

Increased lysosomal stress responses (LSR) are commonly implicated in the pathogenesis of neurodegenerative disorders including HIV-1-associated neurocognitive disorders (HAND). The HIV-1 envelope glycoprotein gp120 causes LSR, increases levels of ferrous iron (Fe²⁺) in the cytosol and in mitochondria, disrupts the reactive species interactome (RSI), and increases neural cell death. Using SH-SY5Y human neuroblastoma and U87MG human astrocytoma cells treated with gp120 and pharmacological modulators, we evaluated redox signaling and LSR by redox-sensitive fluorescent probes, spinning-disk confocal microscopy, flow cytometry, Western blotting, and immunostaining. Here, we report that TRPML1, an endolysosome redox-sensitive cation channel, is mechanistically involved in gp120-induced neurotoxicity. TRPML1 was activated by gp120-induced increases in cytosolic reactive oxygen species (ROS) and resulted in release of Fe²⁺ from endolysosomes in levels sufficient to increase cytosolic levels of Fe²⁺ and ROS as well as decrease levels of hydrogen sulfide (H₂S). Reduced glutathione normally buffers intracellular Fe²⁺, but gp120 decreased endolysosome glutathione levels and disrupted this regulatory control mechanism thereby promoting TRPML1-mediated Fe²⁺ efflux from endolysosomes. TRPML1 redox activation led to changes to the RSI in endolysosomes including increased ROS, lipid peroxidation, nitric oxide, and sulfane sulfur as well as decreased H₂S. These changes were accompanied by increased cysteine oxidation of luminal proteins and endolysosome deacidification. Pharmacological inhibition of TRPML1 or knocking down expression levels of TRPML prevented these effects. Our findings suggest that TRPML1 redox activation controls gp120-induced endolysosome dysfunction and iron/redox imbalance, and further implicates TRPML1 in the pathogenesis of HAND.

Increased lysosomal stress responses (LSR) are commonly implicated in the pathogenesis of neurodegenerative disorders including HIV-1-associated neurocognitive disorders (HAND). The HIV-1 envelope glycoprotein gp120 causes LSR, increases levels of ferrous iron (Fe ²⁺ ) in the cytosol and in mitochondria, disrupts the reactive species interactome (RSI), and increases neural cell death. Here, we report that TRPML1, an endolysosome redox-sensitive cation channel, is mechanistically involved in gp120-induced neurotoxicity. TRPML1 was activated by gp120-induced increases in cytosolic reactive oxygen species (ROS) and resulted in release of Fe ²⁺ from endolysosomes in levels sufficient to increase cytosolic levels of Fe ²⁺ and ROS as well as decrease levels of hydrogen sulfide (H ₂ S). Reduced glutathione normally buffers intracellular Fe ²⁺ , but gp120 decreased endolysosome glutathione levels and disrupted this regulatory control mechanism thereby promoting TRPML1-mediated Fe ²⁺ efflux from endolysosomes. TRPML1 redox activation led to changes to the RSI in endolysosomes including increased ROS, lipid peroxidation, nitric oxide, and sulfane sulfur as well as decreased H ₂ S. These changes were accompanied by increased cysteine oxidation of luminal proteins and endolysosome deacidification. Pharmacological inhibition of TRPML1 or knocking down expression levels of TRPML prevented these effects. Thus, our findings suggest that TRPML1 redox activation controls gp120-induced endolysosome dysfunction and iron/redox imbalance, and further implicates TRPML1 in the pathogenesis of HAND.

Iron continues to be linked to the pathogenesis of neurodegenerative disorders including HIV-1 associated neurocognitive disorders (HAND). People with HIV-1 who use opioids have a higher risk of developing HAND, and HIV-1 proteins and opioids disrupt endolysosome iron homeostasis, increase reactive oxygen species (ROS), and cause neural cell death. Endolysosomes are subcellular acidic organelles that regulate iron metabolism and redox homeostasis. HIV-1 gp120 and opioids induce endolysosome iron release, increasing cytosolic and in mitochondrial iron and ROS and inducing neurotoxicity. However, ROS represent only part of the reactive species interactome (RSI) and little is known about the extent to which HIV-1 proteins and opioids affect the RSI. In SH-SY5Y and U87MG cells, HIV-1 gp120, morphine, and iron supplementation de-acidified endolysosomes, decreased endolysosome Fe²⁺ and H₂S, and increased ROS, lipid peroxidation (LPO) and NO. These changes were accompanied by increased cytosolic and mitochondrial Fe²⁺, ROS, LPO, and NO, decreased H₂S, and increased cell death. All effects were blocked by the endolysosome-specific iron chelator deferoxamine. Endolysosome iron dyshomeostasis induced by HIV-1 gp120, morphine and iron supplementation disrupts inter-organellar iron signaling and RSI homeostasis. Targeting endolysosome iron may mitigate neurotoxicity in HAND and other disorders associated with iron overload and redox imbalance.

Aurora kinase A (AURKA) is a serine/threonine-protein kinase that regulates microtubule organization during neuron migration and neurite formation. Decreased activity of AURKA was found in Alzheimer’s disease (AD) brain samples, but little is known about the role of AURKA in AD pathogenesis. Here, we demonstrate that AURKA is expressed in primary cultured rat neurons, neurons from adult mouse brains, and neurons in postmortem human AD brains. AURKA phosphorylation, which positively correlates with its activity, is reduced in human AD brains. In SH-SY5Y cells, pharmacological activation of AURKA increased AURKA phosphorylation, acidified endolysosomes, decreased the activity of amyloid beta protein (Aβ) generating enzyme β-site amyloid precursor protein cleaving enzyme (BACE-1), increased the activity of the Aβ degrading enzyme cathepsin D, and decreased the intracellular and secreted levels of Aβ. Conversely, pharmacological inhibition of AURKA decreased AURKA phosphorylation, de-acidified endolysosomes, decreased the activity of cathepsin D, and increased intracellular and secreted levels of Aβ. Thus, reduced AURKA activity in AD may contribute to the development of intraneuronal accumulations of Aβ and extracellular amyloid plaque formation.

Human endogenous retroviruses (HERVs) are associated with the pathogenesis of amyotrophic lateral sclerosis (ALS); a disease characterized by motor neuron degeneration and cell death. The HERV-K subtype HML-2 envelope protein (HERV-K Env) is expressed in the brain, spinal cord, and cerebrospinal fluid of people living with ALS and through CD98 receptor-linked interactions causes neurodegeneration. HERV-K Env-induced increases in oxidative stress are implicated in the pathogenesis of ALS, and ferrous iron (Fe ²⁺ ) generates reactive oxygen species (ROS). Endolysosome stores of Fe ²⁺ are central to iron trafficking and endolysosome deacidification releases Fe ²⁺ into the cytoplasm. Because HERV-K Env is an arginine-rich protein that is likely endocytosed and arginine is a pH-elevating amino acid, it is important to determine HERV-K Env effects on endolysosome pH and whether HERV-K Env-induced neurotoxicity is downstream of Fe ²⁺ released from endolysosomes. Here, we showed using SH-SY5Y human neuroblastoma cells and primary cultures of human cortical neurons (HCNs, information on age and sex was not available) that HERV-K Env (1) is endocytosed via CD98 receptors, (2) concentration dependently deacidified endolysosomes, (3) decreased endolysosome Fe ²⁺ concentrations, (4) increased cytosolic and mitochondrial Fe ²⁺ and ROS levels, (5) depolarized mitochondrial membrane potential, and (6) induced cell death, effects blocked by an antibody against the CD98 receptor and by the endolysosome iron chelator deferoxamine. Thus, HERV-K Env-induced increases in cytosolic and mitochondrial Fe ²⁺ and ROS as well as cell death appear to be mechanistically caused by HERV-K Env endocytosis, endolysosome deacidification, and endolysosome Fe ²⁺ efflux into the cytoplasm.

Objectives: Approximately 75 % of marketed drugs have the physicochemical property of being weak bases. Weak-base drugs with relatively high pK_a values enter acidic organelles including endosomes and lysosomes (endolysosomes), reside in and de-acidify endolysosomes, and induce cytotoxicity. Divalent cations within endolysosomes, including iron, are released upon endolysosome de-acidification. Endolysosomes are “master regulators of iron homeostasis”, and neurodegeneration is linked to ferrous iron (Fe²⁺)-induced reactive oxygen species (ROS) generation via Fenton chemistry. Because endolysosome de-acidification-induced lysosome-stress responses release endolysosome Fe²⁺, it was crucial to determine the mechanisms by which a functionally and structurally diverse group of weak base drugs including atropine, azithromycin, fluoxetine, metoprolol, and tamoxifen influence endolysosomes and cause cell death. Methods: Using U87MG astrocytoma and SH-SY5Y neuroblastoma cells, we conducted concentration-response relationships for 5 weak-base drugs to determine EC₅₀ values. From these curves, we chose pharmacologically and therapeutically relevant concentrations to determine if weak-base drugs induced lysosome-stress responses by de-acidifying endolysosomes, releasing endolysosome Fe²⁺ in sufficient levels to increase cytosolic and mitochondria Fe²⁺ and ROS levels and cell death. Results: Atropine (anticholinergic), azithromycin (antibiotic), fluoxetine (antidepressant), metoprolol (beta-adrenergic), and tamoxifen (anti-estrogen) at pharmacologically and therapeutically relevant concentrations (1) de-acidified endolysosomes, (2) decreased Fe²⁺ levels in endolysosomes, (3) increased Fe²⁺ and ROS levels in cytosol and mitochondria, (4) induced mitochondrial membrane potential depolarization, and (5) increased cell death; effects prevented by the endocytosed iron-chelator deferoxamine. Conclusions: Weak-base pharmaceuticals induce lysosome-stress responses that may affect their safety profiles; a better understanding of weak-base drugs on Fe²⁺ interorganellar signaling may improve pharmacotherapeutics.

ID 25123 Poster Board 549 Altered iron metabolism is implicated in many neurodegenerative disorders including HIV-1-associated neurocognitive disorders (HAND). Implicated in the pathogenesis of HAND is the HIV-1 coat protein gp120 as well as comorbid substance abuse, which induce iron dysregulation and the formation of reactive species that cause neuronal death. Because HIV-1 proteins and the ingestion of substances of abuse induce oxidative stress, drug-abusing patients are at a greater risk of oxidative stress-induced neuronal injury and HAND. Previous research has studied the individual effects of gp120- and morphine-induced neurotoxicity with a focus on the formation of reactive oxygen species (ROS); however, their combined neurotoxicity and effects on the newly defined reactive species interactome (RSI) remain largely unknown. Endolysosomes are subcellular acidic organelles known for their function in regulating cellular iron metabolism and redox signaling. Recently, we showed that HIV-1 gp120 and morphine cause endolysosome iron dyshomeostasis, yet the impact of impaired endolysosome iron balance on the RSI remains unknown. Here using SH-SY5Y neuroblastoma and U87MG human glioblastoma cells, we found that HIV-1 gp120 and morphine (1) de-acidified endolysosomes, (2) decreased endolysosome Fe²⁺ levels, (3) increased cytosolic and mitochondrial Fe²⁺ levels, (4) increased endolysosome ROS, lipid peroxidation (LPO) and nitric oxide (NO) levels and decreased endolysosome hydrogen sulfide (H₂S) levels, (5) increased cytosolic ROS levels and decreased cytosolic H₂S levels, (6) increased mitochondrial ROS and LPO, and NO levels and decreased mitochondrial H₂S levels and (7) induced cell death; effects blocked by the endolysosome-specific iron chelator deferoxamine. Thus, an increased understanding of the endolysosome Fe²⁺ pool that plays a critical role in regulating inter-organellar iron signaling, in RSI homeostasis, and in contributing to HIV-1 gp120 and morphine neurotoxicity may provide new insight into potential therapeutic targets for HAND progression. Support/Funding Information: [P20GM139759, P30GM100329, U54GM115458, R01MH100972, R01MH105329, R01MH119000, 2R01NS065957, 2R01DA032444]

Objectives: Opioids including morphine and DAMGO activate mu-opioid receptors (MOR), increase intracellular reactive oxygen species (ROS) levels, and induce cell death. Ferrous iron (Fe²⁺) through Fenton-like chemistry increases ROS levels and endolysosomes are “master regulators of iron metabolism” and contain readily-releasable Fe²⁺stores. However, mechanisms underlying opioid-induced changes in endolysosome iron homeostasis and downstream-signaling events remain unclear.Methods: We used SH-SY5Y neuroblastoma cells, flow cytometry, and confocal microscopy to measure Fe²⁺and ROS levels and cell death.Results: Morphine and DAMGO de-acidified endolysosomes, decreased endolysosome Fe²⁺levels, increased cytosol and mitochondria Fe²⁺and ROS levels, depolarized mitochondrial membrane potential, and induced cell death; effects blocked by the nonselective MOR antagonist naloxone and the selective MOR antagonist β-funaltrexamine (β-FNA). Deferoxamine, an endolysosome-iron chelator, inhibited opioid agonist-induced increases in cytosolic and mitochondrial Fe²⁺and ROS. Opioid-induced efflux of endolysosome Fe²⁺and subsequent Fe²⁺accumulation in mitochondria were blocked by the endolysosome-resident two-pore channel inhibitor NED-19 and the mitochondrial permeability transition pore inhibitor TRO.Conclusions: Opioid agonist-induced increases in cytosolic and mitochondrial Fe²⁺and ROS as well as cell death appear downstream of endolysosome de-acidification and Fe²⁺efflux from the endolysosome iron pool that is sufficient to affect other organelles.

People with human immunodeficiency virus-1 (PLWH) experience high rates of HIV-1-associated neurocognitive disorders (HANDs); clinical symptoms range from being asymptomatic to experiencing HIV-associated dementia. Antiretroviral therapies have effectively prolonged the life expectancy related to PLWH; however, the prevalence of HANDs has increased. Implicated in the pathogenesis of HANDs are two HIV-1 proteins, transactivator of transcription (Tat) and gp120; both are neurotoxic and damage mitochondria. The thread-like morphological features of functional mitochondria become fragmented when levels of reactive oxygen species (ROS) increase, and ROS can be generated via Fenton-like chemistry in the presence of ferrous iron (Fe²⁺). Endolysosomes are central to iron trafficking in cells and contain readily releasable Fe²⁺ stores. However, it is unclear whether the endolysosome store is sufficient to account for insult-induced increases in levels of ROS, mitochondrial fragmentation, autophagy, and cell death. Using U87MG astrocytoma and SH-SY5Y neuroblastoma cells, we determined that chloroquine (CQ), Tat, and gp120 all (1) de-acidified endolysosomes, (2) decreased endolysosome numbers and increased endolysosome sizes, (3) increased mitochondrial numbers (fragmentation), (4) increased autophagosome numbers, (5) increased autolysosome numbers, (6) increased mitochondrial fragments within endolysosomes, and (7) increased cell death. These effects were all blocked by the endolysosome-specific iron chelator deferoxamine (DFO). Thus, the endolysosome de-acidification-induced release of endolysosome Fe²⁺ is sufficient to account for inter-organellar signaling events and cell biology consequences of HIV-1 proteins, including mitochondrial fragmentation, autophagy, and cell death.